Fourier transform infrared (FTIR) spectroscopy has been widely used for the analysis of both protein and nucleic acid secondary structure. This is one of the vibration spectroscopy methods that are extremely sensitive to any change in molecular structure. While numerous reports describe how to proceed to analyze protein and deoxyribonucleic acid (DNA) structures using FTIR, reports related to the analyses of ribonucleic acids (RNAs) are few. Nevertheless, RNAs are versatile molecules involved in a multitude of roles in the cell. In this chapter, we present applications of FTIR for the structural analysis of RNA, including the analysis of helical parameters and noncanonical base pairing, often found in RNA. The effect of temperature pretreatment, which has a great impact on RNA folding, will also be discussed.